补肾益精方对外周血干细胞在RCS大鼠视网膜上的募集及睫状神经营养因子表达的影响

doi:10.3760/cma.j.issn.1674-845X.2018.08.005.

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摘要

目的：观察补肾益精方对外周血骨髓间充质干细胞在视网膜变性模型（RCS）大鼠视网膜募集及睫状神经营养因子（CNTF）分泌的影响，探讨补肾益精方治疗视网膜退行性病变的作用机制。方法：实验研究。采用尾静脉注射方式将1×107个GFP标记的骨髓间充质干细胞（GFP-MSCs）注射至3周龄RCS大鼠外周血，然后按随机数字表法分为蒸馏水组及补肾益精方（BSYJ）组，每组39只。注射后第1天开始分别采用蒸馏水及补肾益精方药液灌胃，正常SD大鼠作为对照组常规饲养。给药7 d、14 d及28 d时分别采用视网膜铺片、免疫荧光显微镜观察GFP-MSCs在视网膜的募集情况；实时荧光定量PCR及免疫荧光双染技术检测视网膜表达CNTF的情况；TUNEL法检测视网膜细胞凋亡情况；采用单因素方差进行数据分析。结果：给药7 d、14 d和28 d时，蒸馏水组GFP-MSCs的数目少于BSYJ组，其中给药28 d时，2 组差异有统计学意义（t=2.71，P=0.001）。给药7 d、14 d和28 d时，BSYJ组CNTF表达较蒸馏水组均明显增加，3 组差异有统计学意义（F=5.763，P=0.003；F=3.165，P=0.042；F=4.839，P=0.004）。给药7 d和28 d时，BSYJ组视网膜细胞凋亡率明显少于蒸馏水组，3组差异有统计学意义（F=0.002，P=0.001；F=0.307，P=0.004）。结论：补肾益精方能够促进外周血干细胞在视网膜上的募集以及促进CNTF的表达，这可能是补肾益精方延缓RCS大鼠视网膜细胞凋亡和病变进程的作用机制之一。

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	李雪丽
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	许凯
	陈强
	梁洁
	王晶莹
	梁丽娜

关键词 ：补肾益精方, 骨髓间充质干细胞, 睫状神经营养因子, 视网膜色素变性

Abstract：

Objective: To measure recruitment of bone marrow-derived mesenchymal stem cells from peripheral blood to the retina of royal college of surgeon (RCS) rats and the expression of ciliary neurotrophic factor (CNTF). We also investigated the mechanism by which Bu Shen Yi Jing Fang (BSYJ) could treat retinal degenerative diseases. Methods: This was an experimental study. RCS rats were injected with 1×107 green fluorescent protein- (GFP-) labeled bone marrow-derived mesenchymal stem cells (GFP-MSCs). The injected rats were assigned to a distilled water group or a BSYJ group according to selection from a random numbertable, and 39 rats in each group. Each rat in the respective groups was gavaged with either distilled water or BSYJ solution one day after injection. Normal SD rats were treated routinely as the control group. At 7, 14, and 28 days after treatment, retina flat mounts were prepared to assess the recruitment of GFP-MSCs to the retina. Quantitative polymerase chain reaction (Q-PCR) and immunofluorescence double labeling were used to determine the effect of BSYJ on expression of ciliary neurotrophic factor (CNTF) in the retina. Apoptosis of retinal cells was measured by the TUNEL method. Data were analyzed using ANOVA.
Results: At 7, 14, and 28 days after treatment, the numbers of GFP-MSCs in the distilled water group were less than in the BSYJ group, and the difference was significant at 28 days (t =2.71, P=0.001). At 7, 14, and 28 days after treatment, CNTF mRNA expression in the BSYJ group was significantly higher than in the
distilled water group (F=5.763, P=0.003; F=3.165, P=0.042; F=4.839, P=0.004). At 7 and 28 days after treatment, the apoptosis rate of retinal cells in BSYJ group was significantly less than in the distilled water group (F=0.002, P=0.001; F=0.307, P=0.004). Conclusions: BSYJ improved recruitment of peripheral
blood stem cells to the retina and increased CNTF expression. The recruitment of stem cells by BSYJ may delay progression of retinal cell apoptosis and degeneration in RCS rats.

Key words： Bu Shen Yi Jing Fang marrow-derived mesenchymal stem cells ciliary neurotrophic factor retinitis pigmentosa

收稿日期: 2018-05-13

基金资助:

国家自然科学基金(81473736，81674033）；国家人力资源和社会保障部留学人员科技活动择优项目（2010-94）；中国中医科学院“一带一路”合作专项课题（GH2017-04-02）

通讯作者: 梁丽娜（ORCID：0000-0003-2408-7852），Email：lianglina163@163.com

引用本文:

李雪丽,唐由之,许凯,陈强,梁洁,王晶莹,梁丽娜. 补肾益精方对外周血干细胞在RCS大鼠视网膜上的募集及睫状神经营养因子表达的影响[J]. 中华眼视光学与视觉科学杂志, 2018, 20(8): 475-483. Xueli Li, Youzhi Tang, Kai Xu, Qiang Chen, Jie Liang, Jingying Wang, Lina Liang. Bu Shen Yi Jing Fang Recruitment of Peripheral Blood Stem Cells to the Retina of RCS Rats and Expression of Retinal Ciliary Neurotrophic Factor. Chinese Journal of Optometry Ophthalmology and Visual science, 2018, 20(8): 475-483. DOI: 10.3760/cma.j.issn.1674-845X.2018.08.005.

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